Tumor necrosis factor-α (TNFα, also known as cachectin) and tumor necrosis factor-β(TNFβ, also known as lymphotoxin) are homologous mammalian endogenous secretory proteins capable of inducing a wide variety of effects on a large number of cell types. The great similarities in the structural and functional characteristics of these two cytokines have resulted in their collective description as “TNF”. Complementary cDNA clones encoding TNFα (Pennica et al, Nature, 312:724 (1984); and TNFβ (Gray et al, Nature, 312:721 (1984)) have been isolated, permitting further structural and biological characterization of TNF.
TNF proteins initiate their biological effect on cells by binding to specific TNF receptor (TNFR) proteins expressed on the plasma membrane of a TNF-responsive cell. TNFα and TNFβ were first shown to bind to a common receptor on the human cervical carcinoma cell line ME-180 (Aggarwal et al, Nature, 318:665 (1985)). Estimates of the size of the TNFR determined by affinity labeling studies ranged from 54 to 175 kDa (Creasey et al, Proc. Natl. Acad. Sci. USA, 84:3293 (1987); Stauber et al, J. Biol. Chem., 263:19098 (1988); and Hohmann et al, J. Biol. Chem., 264:14927 (1989)). Although the relationship between these TNFRs of different molecular mass is unclear, Hohmann et al, J. Biol. Chem., 264:14927 (1989)) reported that at least two different cell surface receptors for TNF exist on different cell types. These receptors have an apparent molecular mass of about 75-80 kDa and about 55-60 kDa, respectively. None of the above publications, however, reported the purification to homogeneity of cell surface TNFRs.
In addition to cell surface receptors for TNF, soluble proteins from human urine capable of binding TNF have also been identified (Peetre et al, Eur. J. Haematol., 41:414 (1988); Seckinger et al, J. Exp. Med., 167:1511 (1988); Seckinger et al, J. Biol. Chem., 264:11966 (1989); Seckinger et al, U.K. Patent Publication No. 2,218,101; and Engelmann et al, J. Biol. Chem., 264:11974 (1989)). The relationship of the above soluble urinary binding proteins was further elucidated when the identification and purification of a second distinct soluble urinary TNF binding protein was reported by Engelmann et al, J. Biol. Chem., 265:1531 (1990). The two urinary proteins disclosed by U.K. Patent Publication No. 2,218,101 and the Engelmann et al publications were shown to be immunochemically related to two apparently distinct cell surface proteins by the ability of antiserum against the binding proteins to inhibit TNF binding to certain cells.
More recently, the molecular cloning and expression of a human 55 kDa TNFR (TNFR-I) has been reported (Loetscher et al, Cell, 61:351 (1990); Schall et al, Cell, 61:361 (1990); and Nophar et al, EMBO J., 9:3269-3278 (1990)). The TNFR of both groups has an N-terminal amino acid sequence which corresponds to the partial amino acid sequence of the urinary binding protein disclosed by U.K. Patent Publication No. 2,218,101; Engelmann et al (1989), supra; and Engelmann et al (1990), supra.
In addition, the molecular cloning and expression of a human 75 kDa TNFR (TNFR-II) has been reported (Smith et al, U.S. Pat. No. 5,395,760; Smith European Patent Publication No. 418014; Smith et al, Science, 248:1019-1023 (1990); Dembic et al, Cytokine, 2:231-237 (1990); and Kohno et al, Proc. Natl. Acad. Sci., USA, 87:8331-8335 (1990).
Smith et al, U.S. Pat. No. 5,395,760 and European Patent Publication No. 418014; as well as Wallach et al, U.S. Pat. No. 5,478,925, disclose multimeric forms of TNFR having enhanced binding affinity for TNF. For example, Smith et al, U.S. Pat. No. 5,395,760 and European Patent Publication No. 418014, disclose a multimeric form of TNFR where one TNFR molecule is linked to another TNFR molecule by a peptide linker (diTNFR), as well as the recombinant production of the same by expressing a gene encoding diTNFR in a transformed host cell.
A particular dimeric form of TNFR is described in Smith et al, U.S. Pat. No. 5,395,760 and European Patent Publication No. 418014, wherein TNFR sequences are substituted for the variable domains of either or both of the immunoglobulin molecule heavy and light chains and having unmodified constant region domains. For example, chimeric TNFR/IgG1 is described which is produced recombinantly using either or both of two chimeric genes——a TNFR/human k light chain chimera (TNFR/Ck) and a TNFR/human γ1 heavy chain chimera (TNFR/Cγ−1). Following transcription and translation of the either chimeric gene in a transformed host, the gene products assemble into a single chimeric antibody molecule having TNFR displayed bivalently.
Jacobs et al, U.S. Pat. No. 5,605,690; Lauffer et al, European Patent Publication No. 464533; Brockhaus et al, European Patent Publication No. 417563; Brockhaus et al, U.S. Pat. No. 5,610,279; Beutler et al, U.S. Pat. No. 5,447,851; Loetscher et al, J. Biol. Chem., 266(27):18324-18329 (1991), Lesslauer et al, Eur. J. Immunol., 21:2883-2886 (1991); Peppel et al, J. Exp. Med., 174(6):1483-1489 (1991); and Mohler et al, J. Immunol., 151:1548-1561 (1993) each disclose chimeric antibodies, wherein an extracellular domain of TNFR is fused to all of the domains of the constant region of a human immunoglobulin heavy chain other than the first domain of said constant region (hereinafter “TNFR:Fc”; or also sometimes referred to in the art as “TNFR-IgG”).
TNFR:Fc is useful, inter alia, in diagnostic assays for TNF, as well as in raising antibodies to TNFR for use in diagnosis and therapy. TNFR:Fc is also useful for suppressing TNF-dependent inflammatory responses or diseases in humans, i.e., to bind or scavenge TNF, thereby providing a means for regulating the immune activities of this cytokine. TNF-dependent inflammatory responses or diseases include arthritis, cachexia, endotoxin shock, hypercalcemia, malignancy, inflammatory bowel disease, osteoporosis, endometriosis, myelodysplastic syndrome, and graft vs. host disease. TNFR:Fc is also useful for treatment of insulin and non-insulin dependent diabetes, HIV infection, asthma, multiple sclerosis and congestive heart failure.
The advantage of mammalian expression systems for TNFR:Fc over bacteria and yeast is mammalian secretory pathways facilitate the assembly, folding and production of biologically active proteins. However, as shown in the Examples below, misfolded TNFR:Fc has been found in TNFR:Fc preparations. That is, TNFR:Fc is resolved by hydrophobic interaction chromatography (HIC) into three peaks (FIG. 4). Peak 1 represents truncated forms arising from proteolytic cleavage. Peak 2 consists of highly pure and biologically active TNFR:Fc, while peak 3 is heterogeneous and is comprised of misfolded product along with other process related impurities (FIG. 5). The misfolded TNFR:Fc, which is formed early in the cell culture process, is transported, and represents a significant proportion (about 25-50%) of the expression product. Such misfolded TNFR:Fc is not preferred when TNFR:Fc is used in any of the above-noted therapies. The present invention was developed in view of the discovery of said misfolded TNFR:Fc, and in order to reduce the production of said misfolded TNFR:Fc.